Vesicular glutamate transporters (VGLUTs) 1 and 2 are expressed by neurons generally accepted to release glutamate as a neurotransmitter, whereas VGLUT3 appears in populations usually associated with a different classical transmitter. We now demonstrate VGLUT2 as well as the vesicular GABA transporter (VGAT) in a subset of presynaptic terminals in the dentate gyrus of the rat hippocampal formation. The terminals are distributed in a characteristic band overlapping with the outer part of the granule cell layer and the inner zone of the molecular layer. Within the terminals, which make asymmetric as well as symmetric synapses onto the somatodendritic compartment of the dentate granule cells, the 2 transporters localize to distinct populations of synaptic vesicles. Moreover, the axons forming these terminals originate in the supramammillary nucleus (SuM). Our data reconcile previous apparently conflicting reports on the physiology of the dentate afferents from SuM and demonstrate that both glutamate and GABA may be released from a single nerve terminal.

The entorhinal cortex (EC) conveys information to hippocampal field CA1 either directly by way of projections from principal neurons in layer III, or indirectly by axons from layer II via the dentate gyrus, CA3, and Schaffer collaterals. These two pathways differentially influence activity in CA1, yet conclusive evidence is lacking whether and to what extent they converge onto single CA1 neurons. Presently we studied such convergence. Different neuroanatomical tracers injected into layer III of EC and into CA3, respectively, tagged simultaneously the direct entorhino-hippocampal fibers and the indirect innervation of CA1 neurons by Schaffer collaterals. In slices of fixed brains we intracellularly filled CA1 pyramidal cells and interneurons in stratum lacunosum-moleculare (LM) and stratum radiatum (SR). Sections of these slices were scanned in a confocal laser scanning microscope. 3D-reconstruction was used to determine whether boutons of the labeled input fibers were in contact with the intracellularly filled neurons. We analyzed 12 pyramidal neurons and 21 interneurons. Perforant path innervation to pyramidal neurons in our material was observed to be denser than that from CA3. All pyramidal neurons and 17 of the interneurons received contacts of both perforant pathway and Schaffer input on their dendrites and cell bodies. Four interneurons, which were completely embedded in LM, received only labeled perforant pathway input. Thus, we found convergence of both projection systems on single CA1 pyramidal and interneurons with dendrites that access the layers where perforant pathway fibers and Schaffer collaterals end.

Presynaptic boutons and associated postsynaptic structures in the CNS express markers that are highly synapse type-specific. In multilabel immunofluorescence imaging, coexpression of such markers appears as overlap of signals in the same structures whereas closely related yet segregated markers, e.g., located pre-and postsynaptically, translate into signals that touch. 'Overlap' and 'touch' occur in three dimensions (3D). The instrument of choice to study overlap vs. touch of small objects in tissue volumes is the confocal laser scanning microscope (CSLM). To quantify overlap and touch we used two paradigms. Overlap was studied in rat brain sections triple-immunostained with antibodies against markers predominantly located presynaptically: glutamic acid decarboxylase, vesicular glutamate transporter 2, and calretinin. Touch was studied in rat temporal cortex where afferent, tracer-labeled entorhinohippocampal fibers in the subiculum were imaged together with possible postsynaptic target neurons immunostained with an antibody against the calcium binding protein, parvalbumin. Z-series of CLSM images were obtained in multiple channels. After post-acquisition deconvolution we further processed the images via software written in the C/C++ SCIL Image computer programming environment. The software receives parameters via scripts, after which it first identifies 3D objects through establishing isodensity envelopes around pixels representing small biologic structures (in our case: boutons) and then compares associated Z-series in which it determines whether there is overlap or touch between recognized 3D objects. Finally, graphic and numeric output is produced. With this script-commanded software we feel equipped to accurately and objectively quantify overlap and touch.

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The entorhinal cortex of the rat (EC) contains a dense fiber plexus that expresses the calcium-binding protein calretinin (CR). Some CR fibers contain vesicular glutamate transporter 2 (VGluT2, associated with glutamatergic neurotransmission). CR-VGluT2 coexpressing fibers may have an extrinsic origin, for instance, the midline thalamic nucleus reuniens. Alternatively, they may belong to cortical interneurons. We studied the first possibility with anterograde and retrograde neuroanatomical tracing methods combined with CR and VGluT2 immunofluorescence and confocal laser scanning. The alternative possibility was studied with in situ hybridization fluorescence histochemistry for VGluT2 mRNA combined with CR immunofluorescence. In the anterograde tracing experiments, we observed many labeled reuniens fibers in EC expressing CR. Some of these labeled fibers contained immunoreactivity for VGluT2 and CR. In the complementary retrograde tracing experiments, we found retrogradely labeled cell bodies in nucleus reuniens of the thalamus that coexpressed CR. We also examined the colocalization of VGluT2 and CR in the entorhinal cortex by using in situ hybridization and CR immunofluorescence. In these experiments, we observed CR-immunopositive cortical neurons that coexpressed VGluT2. For the same sections, with CR as the principal marker and parvalbumin as a control marker, we found that parvalbumin neurons were negative for VGluT2 mRNA. Thus, CR-VGluT2-expressing axon terminals in EC belong to two sources: projection fibers from the thalamus and axon collaterals of local interneurons. VGluT2 expression is linked to the synaptic transmission of the excitatory neurotransmitter glutamate, so these thalamic CR-VGluT2 projection neurons and entorhinal CR-VGluT2 interneurons should be regarded as excitatory.